A variety of cellular processes are mediated or modified by increases in the cytosolic calcium concentration ([Ca]i). Organic chelators (cages) of Ca2+ are commercially available that release Ca2+ after absorption of an ultraviolet photon, and these agents can be loaded into the cytosol of cells to provide a means for rapid modification of [Ca]i. Experiments that investigate the differential response of cells to calcium released at distinct three-dimensional coordinates require an ability to excite cages with high resolution in the x-y plane of the stage and along the axis of the excitation beam. We have demonstrated two-photon calcium uncaging and have determined the two photon action cross sections of three calcium cages; Azid-1, DM-Nitrophen, and NPEGTA, with peak action cross sections of 3 GM, 0.02 GM, and <0.008 GM, respectively.